DNA sequences coding for a mammalian glucuronyl C5-epimerase and a process for its production

ABSTRACT

An isolated or recombinant DNA sequence coding for a mammalian, including human, glucuronyl C5-epimarase or a functional derivative thereof capable of converting D-glucuronic acid (GlcA) to L-iduronic acid (IdoA); a recombinant expression vector comprising such DNA sequence; a host cell transformed with such recombinant expression vector; a process for the manufacture of a glucuronyl C5-epimerase or functional derivative thereof capable of converting GlcA to IdoA, comprising cultivation of a cell-line transformed with such recombinant expression vector; and a glucuronyl C5-epimerase or functional derivative thereof prepared by such process.

[0001] The present invention relates to an isolated or recombinant DNAsequence coding for a glucuronyl C5-epimerase capable of convertingD-glucuronic acid to L-iduronic acid. The invention also relates to aprocess for the manufacture of such epimerase.

BACKGROUND OF THE INVENTION

[0002] Heparin and heparan sulfate are complex, sulfatedglycosaminoglycans composed of alternating glucosamine and hexuronicacid residues. The two polysaccharides are structurally related butdiffer in composition, such that heparin is more heavily sulfated andshows a higher ratio of L-iduronic acid (IdoA)/D-glucuronic acid (GlcA)units. (Kjellén, L. and Lindahl, U. (1991) Annual Review of Biochemistry60, 443-475; Salmivirta, M., Lidholt, K. and Lindahl, U. (1996) TheFASEB Journal 10, 1270-1279). Heparin is mainly produced by connectivetissue-type mast cells, whereas heparan sulfate has a ubiquitousdistribution and appears to be expressed by most cell types. Thebiological roles of heparin and heparan sulfate are presumably largelydue to interactions of the polysaccharides with proteins, such asenzymes, enzyme inhibitors, extracellular-matrix proteins, growthfactors/cytokines and others (Salmivirta, M., Lidholt, K. and Lindahl,U. (1996) The FASEB Journal 10, 1270-1279). The ineractions tend to bemore or less selective/specific with regard to carbohydrate structure,and thus depend on the amounts and distribution of the various sulfategroups and hexuronic acid units. Notably, IdoA units are believed togenerally promote binding of heparin and heparan sulfate chains toproteins, due to the marked conformational flexibility of these residues(Casu, B., Petitou, M., Provasoli, M. and Sinay, P. (1988) Trends inBiochemical Sciences 13, 221-225).

[0003] Heparin and heparan sulfate are synthesized as proteoglycans. Theprocess is initiated by glycosylation reactions that generate saccharidesequences composed of alternating GlcA and N-acetylglucosamine (GlcNAc)units covalently bound to peptide core structures. The resulting(GlcAβ1,4-GlcNAca1,4-)_(n) disaccharide repeats are modified, probablyalong with chain elongation, by a series of enzymatic reactions that isinitiated by N-deacetylation and N-sulfation of GlcNAc units, continuesthrough C-5 epimerization of GlcA to IdoA residues, and is concluded bythe incorporation of O-sulfate groups at various positions. TheN-deacetylation/N-sulfation step has a key role in determining theoverall extent of modification of the polymer chain, since the GlcA C-5epimerase as well as the various O-sulfotransferases all depend on thepresence of N-sulfate groups for substrate recognition. While the GlcNAcN-deacetylation and N-sulfation reactions are both catalyzed by the sameprotein, isolation and molecular cloning ofN-deacetylase/N-sulfotransferase from different tissue sourcesimplicated two distinct forms of the enzyme. The two enzyme types differwith regard to kinetic properties, and it has been suggested that theymay be differentially involved in the biosynthesis of heparin andheparan sulfate.

SUMMARY OF THE INVENTION

[0004] The present invention provides for an isolated or recombinantDNA-sequence coding for a mammalian, including human, glucuronyl C-5epimerase or a functional derivative thereof capable of convertingD-glucuronic acid (GicA) to L-iduronic acid (IdoA).

[0005] The invention also provides for a recombinant expression vectorcontaining a transcription unit comprising a DNA sequence as describedabove, a transcriptional promoter, and a polyadenylation sequence.

[0006] The invention also provides for a process for the manufacture ofa glucuronyl C-5 epimerase or a functional derivative thereof capable ofconverting D-glucuronic acid (GlcA) to L-iduronic acid (IdoA),comprising cultivation of a cell line transformed with the aboverecombinant expression vector in a nutrient medium allowing expressionand secretion of said epimerase or functional derivative thereof.

[0007] Specific DNA sequences according to the invention are defined inappended claims 2, 3 and 4.

[0008] Furthermore, the invention provides for a host cell transformedwith such recombinant expression vector.

[0009] Finally, the invention covers a glucuronyl C-5 epimerase or afunctional derivative thereof whenever prepared by the process outlinedabove.

BRIEF DESCRIPTION OF THE APPENDED FIGURES AND SEQUENCE LISTING

[0010] Sequence listing: Nucleotide sequence and the predicted aminoacid sequence of the C5-epimerase. The predicted amino acid sequence isshown below the nucleotide sequence. The numbers on the right indicatethe nucleotide residue and the amino acid residue in the respectivesequence. The five sequenced peptides appear in bold. The N-terminalsequence of the purified protein is shown in bold and italics. Thepotential N-glycosylation sites (*) are shown. The potentialtransmembrane region is underlined.

[0011]FIG. 1. In vitro transcription-translation. The epimerase cDNA wasinserted into a pcDNA3 expression vector and linearized with XbaI at the3′-end. It was then subjected to in vitro transcription-translation in arabbit reticulocyte lysate system in the presence of [³⁵S]methionine, asdescribed in “Experimental Procedures”. The translation product ofepimerase cDNA (Epi) has a molecular weight of ˜50 kDa, by comparisonwith the LMW protein standard. A 118 kDa control sample ofβ-galactosidase (C), expressed in the same system, is shown forcomparison.

[0012]FIG. 2. Effect of the expressed C5-epimerase on N-deacetylated,N-sulfated capsular polysaccharide from E. coli K5. Metabolically³H-labeled K5 polysaccharide was N-deacetylated and N-sulfated, and wasthen incubated with (A) lysate of Sf9 cells infected with recombinantC5-epimerase; (B) lysate of Sf9 cells infected with recombinantβ-glucuronidase. The incubation products were treated with HNO₂/NaBH₄,and the resultant hexuronyl-anhydromannitol disaccharides were recoveredand separated by paper chromatography. The arrowheads indicate themigration positions of glucuronosyl-anhydromannitol (GM) andiduronosyl-anhydromannitol (IM) disaccharide standards. For furtherinformation see “Experimental Procedures”.

[0013]FIG. 3. Northern analysis of C5-epimerase mRNA expressed in bovinelung and mastocytoma cells. Total RNA from each tissue/cell line wasseparated by agarose gel electrophoresis. A blot was prepared, probedwith a ³²P-labeled 2460-bp fragment of the epimerase cDNA clone, andfinally exposed to X-ray film. (Kodak, Amersham). The arrow indicatesthe positions of molecular standards. For further information see“Experimental Procedures”.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to DNA sequences coding for amammalian glucuronyl C5-epimerase or a functional derivative thereof,such epimerase or derivative being capable of converting D-glucuronicacid (GlcA) to L-iduronic acid (IdoA). The term “mammalian” is intendedto include also human varieties of the enzyme.

[0015] As used herein the definition “glucuronyl C5-epimerase or afunctional derivative thereof” refers to enzymes which have thecapability of converting D-glucuronic acid to L-iduronic acid.Accordingly, the definition embraces all epimerases having suchcapability including functional variants, such as functional fragments,mutants resulting from mutageneses or other recombinant techniques.Furthermore, the definition is intended to include glycosylated orunglycosylated mammalian glucuronyl C5-epimerases, polymorfic or allelicvariants and other isoforms of the enzyme. “Functional derivatives” ofthe enzyme can include functional fragments, functional fusion proteinsor functional mutant proteins. Such epimerases included in the presentinvention can have a deletion of one or more amino acids, such deletionbeing an N-terminal, C-terminal or internal deletion. Also truncatedforms are envisioned as long as they have the conversion capabilityindicated herein.

[0016] Operable fragments, mutants or truncated forms can suitably beidentified by screening. This is made possible by deletion of forexample N-terminal, C-terminal or internal regions of the protein in astep-wise fashion, and the resulting derivative can be analyzed withregard to its capability of the desired conversion of D-glucuronic acidto L-iduronic acid. If the derivative in question operates in thiscapacity it is considered to constitute a functional derivative of theepimerase proper.

[0017] Examples of useful epimerases are proteins having the sequence asshown in the sequence listing or substantially as shown in the sequencelisting and functional portions thereof.

[0018] Experimental Procedures

[0019] Peptide Purification and Sequencing—The 52 kDa epimerase protein(˜1 μg), purified from a detergent extract of bovine liver bychromatography on O-desulfated heparin-Sepharose, Red-Sepharose,Phenyl-Sepharose, and Concanavalin. A-Sepharose (Campbell, P.,Hannesseon, H. H., Sandback, D., Rodén, L., Lindahl, U. and Li, J.-p.(1994) J Biol Chem 269, 26953-26958), was subjected to direct N-terminalsequencing using a model 470A protein sequenator (Applied Biosystems)equipped with an on-line 120 phenylthiohydantoin analyzer (Tempst, P.,and Riviere, L. (1989) Anal. Biochem. 183, 290-300). Another sample (˜1μg) was applied to preparative (12%) SDS-PAGE and was then transferredto a PVDF membrane. After staining the membrane with Coomassie Blue, theenzyme band was excised. Half of the material was submitted to directN-terminal sequence analysis, whereas the remainder was digested withLys-C (0.0075 U; Waco) in the presence of 1% RTX-100/10%acetonitrile/100 mM Tris-HCl, pH 8.0. The generated peptides wereseparated on a reverse phase C4-column, eluted at a flow rate of 100μl/min with a 6-ml 10-70% acetonitrile gradient in 0.1% trifluoroaceticacid, and detected with a 990 Waters diode-array detector. Selectedpeptides were then subjected to sequence analysis as described above.

[0020] Probes for Screening—Total RNA was extracted from bovine liveraccording to the procedures of Sambrook et al. (1989). Single-strandedcDNA was synthesized by incubating ˜5 μg of bovine liver total RNA(denatured at 65° C., 3 min) with a reaction mixture containing 1 unitRNAse inhibitor (Perkin-Elmer Corp.), 1 mM of each dNTP, 5 μM randomnucleotide hexamer and 1.25 units of murine leukemia virus reversetranscriptase (Perkin-Elmer Corp.) in a buffer of 10 mM Tris-HCl, pH8.3. The mixture was kept at 42° C. for 45 min and then at 95° C. for 5min. Degenerated oligonucleotide primers were designed based on theamino-acid sequence determined for one of the internal peptides derivedfrom the purified epimerase (Table I). Single-stranded bovine liver cDNAwas applied to PCR together with 100 pmols of primers 1 (sense) and 3(antisense), in a total volume of 100 μl containing 1 μl of 10% Tween20, 6 mM MgCl₂, 1 μM of each dNTP, and 2.5 units Taq polymerase(Pharmacia Biotech) in a buffer of 10 mM Tris-HCl, pH 9.0. The reactionproducts were separated on a 12% polyacrylamide gel. A ˜100-bp band wascut out from the gel and reamplified using the same PCR conditions.After an additional polyacrylamide gel electrophoresis, the product wasisolated and sequenced, yielding a 108-bp sequence. This PCR product wassubcloned into a pUC119 plasmid. The DNA fragment cleaved from theplasmid was labeled with [³²P]dCTP (DuPont NEN) using a Randon PrimedDNA Labeling Kit (Boehringer Mannhem).

[0021] Screening of cDNA Library—A bovine lung cDNA library constructedin a lgt10 vector (Clontech) was screened with the 108-bp PCR fragmentas hybridizing probe. The nitrocellulose replicas of the library plaqueswere prehybridized in 6×SSC, 5×Denhart's solution containing 0.1% SDSand 0.1 mg/ml denatured salmon DNA for 2 hours at 65° C. Hybridizationwas carried out at 42° C. in the same solution containing ³²P-labledprobe for 16-18 hours. The filters were washed two times with 2×SSC,0.5% SDS and two times with 0.5×SSC, 1% SDS at the same temperature. Thelibrary was repeatedly screened twice under the same conditions.Finally, the entire cDNA phage library was subjected to PCRamplification using lgt10 forward and reverse primers (Clontech) with aepimerase cDNA specific primer (5′-GCTGATTCTTTTCTGTC-3′).

[0022] Subcloning and Sequencing of cDNA Inserts—cDNA inserts, isolatedby preparative agarose gel elctrophoresis (Sambrook et al., 1989) afterEcoRI restriction cleavage of recombinant bacteriophage DNA, weresubcloned into a pUC119 plasmid. The complete nucleotide sequence wasdetermined independently on both strands using the dideoxy chaintermination reaction either with [³⁵S]dATP and the modified T7 DNApolymerase (Sequenase version 2.0 DNA Sequencing Kit; U.S. BiochemicalCorp.) or the ALF™ System (Pharmacia Biotech). DNA sequences werecompiled and analyzed using the DNASTAR™ program (Lasergene).

[0023] Polyclonal Antibodies and Immunodetection—A peptide correspondingto residues 77-97 of the deduced epimerase amino-acid sequence waschemically synthesized (Ake Engstrom, Department of Medical andPhysiological Chemistry, Uppsala University, Sweden), and was thenconjugated to ovalbumin using glutaraldehyde (Harlow, E. and Lane, D.(1989) in Antibodies: A Laboratory Manual, pp 78-79, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). A rabbit was immunized with thepeptide conjugates together with Freund's adjuvant. After 6 boosts (eachwith 240 μg conjugated peptide) blood was collected and the serumrecovered. The antibody fraction was further purified on a ProteinA-Sepharose column (Pharmacia Biotech), and used for immunoblotting.

[0024] Samples of GlcA C5-epimerase were separated under denaturingconditions by 12% SDS-PAGE, and were then transferred to anitrocellulose membrane (Hybond™ ECL). ECL immunoblotting was performedaccording to the protocol of the manufacturer (Amersham). Briefly, themembrane was first treated with blocking agent, then incubated withpurified antibody, and finally incubated with the peroxidase labeledanti-rabbit antibody. After adding the ECL reagent, the light emitted bythe chemical reaction was detected by exposure to Hyperfilm™ ECL for30-60 sec.

[0025] Northern Blot Hybridization—Bovine liver and lung total RNA wasprepared according to Sambrook, J., Fritsch, E. F. and Maniatis, T.(1989) in Molecular Cloning: A Laboratory Manual, Cold Spring HaraborLaboratory, Cold Spring Harbor N.Y.), and mouse matocytoma (MCT) totalRNA was extracted from a tumor cell line (Montgomery, R. I., Lidholt,K., Flay, N. W., Liang, J., Verter, B., Lindahl, U. and Esko, J. D.(1992) PNAS 89, 11327-113331) as described by Chomczynski and Sacci(1987;. Total RNA from each tissue (˜20 μg samples) was denatured in 50%formamide (v/v), 5% formaldehyde, 20 mM Mops buffer, pH 7.0, at 65° C.for 5 min. The denatured RNA was separated by electrophoresis in 1.2%agarose gel containing 5% formaldehyde (v/v), and was then transferredto a Hybond N⁺ nylon membrane (Amersham). The RNA blot waspre-hybridized in ExpressHyb Hybridization Solution (Clontech) at 65° C.for 1 h, and subsequently hybridized in the same solution with-a[³²P]dCTP-labeled DNA probe (a 2460 bp fragment including the 5′-end ofthe cDNA clone; see the sequence listing). The membrane was washed in2×SSC, 0.5% SDS at the same temperature for 2×15 min and in 0.5×SSC,0.5% SDS for 2×15 min. The membrane was exposed to a Kodak X-ray film at−70° C. for 24 h.

[0026] In Vitro Translation—The 3-kb GlcA C5-epimerase clone, insertedin a pcDNA3 expression vector (Invitrogen) was linearized at the 3′-endby restriction enzyme XbaI. In vitro translation was carried out with aLinked T7 transcription-translation system (Amersham) α-cording to theinstructions of the manufacturer. The corresponding mRNA generated byincubation of 0.5 μg linearized plasmid DNA with a T7 polymerasetranscription mix (total volume, 10 μl; 30° C.; 15 min) was mixed withan optimized rabbit reticulocyte lysate containing 50 μCi[³⁵S]methionine (total volume, 50 μl), and further incubated at 30° C.for 1 h. A sample (5 μl) of the product was subjected to 12% SDS-PAGE.The gel was directly exposed to a Kodak X-ray film. After exposure, theapplied protein molecular standards (LMW Molecular Calibration Kit,Pharmacia Biotech) were visualized by staining the gel with CoomassieBlue.

[0027] Expression of the GlcA C5-Epimerase—The GlcA C5-epimerase wasexpressed using a BacPAK8™ Baculovirus Expression System (Clontech),according to the instructions by the manufacturer. Two oligonucleotides,one at the 5′-end of the cDNA clone (1-17 bp, sense) and the other atthe 3′-end of the coding sequence (1387-1404 bp, antisense), were usedto PCR amplify the coding sequence of the C5-epimerase cDNA clone. Theresulting fragment was cloned into the BacPAK8 vector. Sf9 insect cells,maintained in Grece's Insect Medium (GibcoBRL) supplemented with 10%fetal calf serum and penicillin/streptomycin, were then cotransfected bythe C5-epimerase construct along with viral DNA. Control transfectionswere performed with constructs of a β-glucuronidase cDNA constructincluded in the expression kit, and a mouse cDNA coding for the GlcNAcN-deacetylase/N-sulfotransferase implicated in heparin biosynthesis(Eriksson, I., Sandbäck, D., Ek, B., Lindahl, U. and Kjellén, K. (1994)J. Biol. Chem. 269, 10438-10443; Cheung, W F., Eriksson, I.,Kusche-Gullberg, M., Lindahl, U. and Kjellén, L. (1996) Biochemistry 35,5250-5256). Single plaques of each co-transfected recombinant werepicked and propagated. Two Petri dishes (60-mm) of Sf9 cells wereinfected by each recombinant virus stock and incubated at 27° C. for 5days. The cells from one dish were used for total RNA extraction andNorthern analysis performed as described above. Cells from the otherdish were lysed in a buffer of 100 mM KCl, 15 mM EDTA, 1% Triton X-100,50 mM HEPES, pH 7.4, containing 1 mM PMSF and 10 μg/ml pepstatin A.Supernatants of cell lysates as well as conditioned media were analyzedfor epimerase activity. Protein contents of the cell lysates wereestimated by the method of Bradford (1976) or by the BCA reagentprocedure (Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K.,Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson,B. J. and Klenk, D. C. (1985) Anal. biochem 150, 76-85).

[0028] Demonstration of GlcA C5-Epimerase Activity—Epimerase activitywas assayed using a biphasic liquid scintillation counting procedure,essentially as described by Campbell et al. (1994) above. The reactionmixtures, total volume 55 μl, contained 25 μl cell lysate or medium, 25μl of 2×epimerase assay buffer (20 mM HEPES, 30 mM EDTA, 0.02% TritonX-100, 200 mM KCl, pH 7.4) and 5 μl of substrate (10,000 cpm ³H). Thesubstrate was a chemically N-deacetylated and N-sulfated polysaccharide,obtained from E. coli K5 according to the procedure of Campbell et al.(1994), except that D-[5-³H]glucose was substituted for D-[1-³H]glucose.

[0029] Enzymatic conversion of D-glucuronic to L-iduronic acid wasdemonstrated using the metabolically 1-³H-labeled substrate(N-deacetylated, N-sulfated capsular polysaccharide from E. coli K5) andthe analytical procedure described by Campbell et al. (1994). A sample(˜20 μg; 200,000 cpm of ³H) of the modified polymer was incubated with250 μl of cell lysate in a total volume of 300 μl epimerase assay bufferat 37° C. for 6 hours. The incubation was terminated by heating at 100°C. for 5 min. The sample was mixed with 50 μg of carrier heparin andreacted with nitrous acid at pH 1.5 (Shively, J., and Conrad, H. E.(1976) Biochemistry 15, 3932-3942), followed by reduction of theproducts with NaBH₄. The resultant hexuronyl-anhydromannitoldisaccharides were recovered by gel chromatography on a column (1×200cm) of Sephadex G-15 in 0.2 M NH₄ HCO₃, lyophilized, and subjected topaper chromatography on Whatman No 3 MM paper in ethyl acetate/aceticacid/water (3:1:1).

[0030] Results

[0031] Generation of a Probe and Screening of cDNA Library—Amino acidsequence data for the ˜52 kDa protein were obtained by digesting highlypurified epimerase with lysine-specific protease, followed by separationof the generated peptides on a reverse phase column. The five mostprominent peptides were isolated and subjected to amino-acid sequencing(Table I). One of the peptides (peptide 1) was found to correspond tothe N-terminal sequence of the native protein. The sequence of thelargest peptide obtained (peptide 5 in Table I), was used to design twosense and one antisense degenerate oligonucleotide primers, as shown inTable I. A DNA probe was produced by PCR using primers 1 and 3 withbovine liver cDNA as template. The resultant ˜100 bp DNA fragment waspurified by polyacrylamide gel electrophoresis, reamplified using thesame primers, and finally isolated by electrophoresis. The identity ofthe product was ascertained by “nested” PCR, using primers 2 and 3,which yielded the expected ˜60 bp fragment (data not shown). Moreover,sequencing of the larger (108 bp) DNA fragment gave a deduced amino-acidsequence identical to that of the isolated peptide (Table I).

[0032] The 108-bp PCR product was labeled with [³²P]dCTP and used forscreening of a bovine lung lgt10 library. One hybridizing clone,containing a 3-kb insert, was identified. Repeated screening of the samelibrary yielded two additional positive clones, both of which were ofsmaller size. Subsequent sequencing showed both of the latter clones tobe contained within the 3.0-kb species (data not shown). The 3-kb clonewas sequenced through both strands and was found to contain altogether3073 bp; an additional 12-bp sequence was added at the 5′-end throughcharacterization of a separate clone obtained by PCR amplification ofthe phage library (see “Experimental Procedures”).

[0033] Characterization of cDNA and Predicted Protein Structure—Thetotal cDNA sequence identified, in all 3085 bp, contains an open readingframe corresponding to 444 amino-acid residues (the sequence listing).Notably, the coding region (1332 bp) is heavily shifted toward the5′-end of the available cDNA, and is flanked toward the 3′-end by alarger (1681 bp) noncoding segment. The deduced amino-acid sequencecorresponds to a 49,905 dalton polypeptide. All of the five peptidesisolated after endo-peptidase digestion (Table I) were recognized in theprimary structure deduced from the cDNA (the sequence listing). One ofthese peptides (peptide 1) is identical to the N-terminus of theisolated liver protein. This peptide was found to match residues 74-86of the deduced polypeptide sequence. The enzyme isolated from bovineliver thus represents a truncated form of the native protein. Generationof mRNA from an expression vector inserted with the 3-kb cDNA clone,followed by incubation of the product with rabbit reticulocyte lysate inthe presence of [³⁵S]methionine, resulted in the formation of a distinctlabeled protein with an estimated M_(r) of ˜50 kDa (FIG. 1). Thisproduct was recognized in immunoblotting (data not shown) by polyclonalantibodies raised against a synthetic peptide corresponding to residues77-97 (see the sequence listing) of the deduced amino-acid sequence. Thesame antibodies also reacted with the isolated ˜52 kDa bovine liverprotein (data not shown). These observations establish that the 3-kbcDNA is derived from the transcript that encodes the isolated ˜52 kDabovine liver protein.

[0034] The cDNA structure indicates the occurrence of 3 potentialN-glycosylation sites (the sequence listing). Sugar substituents may beimportant for the proper folding and catalytic activity of the enzyme,since the protein expressed in bacteria (which also gave a strongWestern signal towards the polyclonal antibodies raised against thesynthetic peptide; data not shown) was devoid of enzymatic activity. Apotential transmembrane region is underlined in the sequence listing.The predicted protein contains two cystein residues, only one of whichα-curs in the isolated (truncated) protein. Since NEM was inhibitory toepimerase activity (data not shown), this single cystein unit may beessential to the catalytic mechanism.

[0035] Functional Expression of the GlcA C5-Epimerase—A variety ofexpression systems were tested in attempts at generating the clonedprotein in catalytically active form. A protein obtained by in vitrotranslation using a rabbit reticulocyte lysate system (see FIG. 1)showed no detectable epimerase activity. A construct made by insertingthe 3-kb cDNA into a pcDNA3 vector (Invitrogen) failed to induce mRNAformation (or translation) in any of the cell lines tested (humanembryonic kidney (293), COS-1 or CHO cells) (data not shown). We alsoattempted to express the enzyme in a bacterial pET system (Novagen). Thetransformed bacteria yielded appreciable amounts of immunoreactiveprotein which, however, lacked detectable enzyme activity (data notshown).

[0036] Cotransfection of epimerase recombinant with baculovirus into Sf9insect cells resulted in the generation of abundant GlcA C5-epimeraseactivity (Table II). In two separate experiments, the lysates from cellsinfected with the same epimerase recombinant virus stock showed >10-foldhigher enzyme activities, on a mg protein basis, than the correspondingfractions from cells infected with control recombinant virus stock. Theconditioned media of cells infected with epimerase recombinant showed20-30-fold higher enzyme activities than the corresponding fractionsfrom cells infected with control plasmid virus stock. Transfections withcDNA encoding other enzymes, such as a β-glucuronidase, or the mousemastocytoma GlcNAc N-deacetylase/N-sulfotransferase involved in heparinbiosynthesis (Eriksson et al., 1994), did not significantly increase theepimerase activity beyond control levels. Notably, the higher ³H₂Orelease recorded for control samples as compared to heat-inactivatedexpressed enzyme (Table II) suggests that the insect cellsconstitutively produce endogenous C5-epimerase.

[0037] The polysaccharide substrate used for routine assays of epimeraseactivity was obtained by chemically N-deacetylating and N-sulfating thecapsular polysaccharide [(GlcAβ1,4-GlcNAca1,4)_(n)] of E. coli K5 thathad been grown in the presence of (5-³H)glucose. The data in Table IIthus reflect the release of ³H₂O from 5-³H-labeled GlcA units in themodified polysaccharide, due to enzyme action (Jacobsson, I., Bäckström,G., Höök, M., Lindahl, U., Feingold, D. S., Malmström, M, and Rodén, L.(1979) J. Biol. Chem. 254, 2975-2982; Jacobsson, I., Lindahl, U.,Jensen, J. W., Rodén, L., Prihar, H. and Feingold, D. S. (1984) Journalof Biological Chemistry 259, 1056-1064). More direct evidence for theactual conversion of GlcA to IdoA residues was obtained by incubatingthe expressed enzyme with an analogous substrate, obtained followingincubation of the bacteria with [1-³H]glucose. This substrate willretain the label through the epimerization reaction, and can thereforebe used to demonstrate the formation of IdoA-containing disaccharideunits. Following incubation with the recombinant epimerase, 21% of thehexuronic acid residues was converted to IdoA, as demonstrated by paperchromatography of disaccharide deamination products (FIG. 2). Thecomposition of the incubated polysaccharide thus approached theequilibrium ratio of IdoA/GlcA, previously determined to ˜3/7¹).

[0038] Northern Analysis—Total RNA, from bovine liver, lung, and mousemastocytoma, were analysed by hybridization with a 2460-bp DNA fragmentfrom epimerase cDNA clone as a probe. Both bovine liver and lung gaveidentical transcription patterns, with a dominant transcript of 9 kb anda weak ˜5 kb band (FIG. 3). By contrast, the mastocytoma RNA showed onlythe 5 kb transcript.

[0039] It is to be noted that the present invention is not restricted tothe specific embodiments of the invention as described herein. Theskilled artisan will easily recognize equivalent embodiments and suchequivalents are intended to be encompassed in the scope of the appendedclaims. TABLE I Peptide and primer sequences A. N-terminal sequences ofisolated C5-epimerase    1. PNDWXVPKGCFMA (free solution)    2.PXDWTVPKGXF (band excised from PVDF-membrane) B. Peptide sequences    1.PNDXTVPK    2. XXIAPETSEGXSLQL    3. GGWPIMVTRK    4. FLSEQHGV    5.KAMLPLYDTGSGTIYDLRHFMLGIAPNLAXWDYHTT       primer 1       primer2       primer 3       (sense)        (sense)        (antisense) C.Primer^(a) Degeneracy    1 (S) 5′-cc gaattcAARGCNATGYT 384 NCCNYT3′^(b)   2 (S) 5′-cc gaattcGAYYTNMGNCAY 288 TTYATG-3′    3 (AS) 5′-ccggatccGTNGTRTGRTA 32 RTCCCA-3′

[0040] TABLE II Expression of HexA C5-epimerase in Sf9 cells Sf9 cells(1 × 10⁶ in 4 ml medium) were seeded in 60-mm Petri dishes and incubatedfor three hours at 27° C. After the cells were attached, the medium wasremoved, and 200 μl of recombinant virus stock was added to infect thecells at room temperature for 1 h. The virus suspension was aspiratedand 4 ml of medium was added to each dish. The cells were incubated at27° C. for 5 days. The medium was transferred into a steril tube andcentrifuged. The cells were collected, washed twice with PBS and lysedwith 300 μl of homogenization buffer as described under “ExperimentalProcedures”. Aliquots (25 μl) of cell lysate and medium were assayed forepimerase activity. The activity is expressed as release of ³H from K5polysaccharide per hour. The data is mean value of three independentassays. Epimerase Activity Cell lysate Medium Construct (cpm/mg/h)(cpm/ml/h) HexA C5-Epimerase-1 102670 ± 5540 45200 ± 1770 HexAC5-Epimerase-2 123270 ± 4660 52610 ± 810 HexA C5-Epimerase-1 240 610(heat-inactivted) N-Deacetylase/  9520 ± 620  1350 ± 280sulfotransferase β-Glucuronidase  8460 ± 1270  1610 ± 440 BacPAK plasmid 5150 ± 880  2820 ± 690 Neo  7250 ± 370  550 ± 120

[0041]

1 13 1 17 DNA Human 1 gctgattctt ttctgtc 17 2 13 PRT Human PEPTIDE(5)..(5) Amino acid 5 is Xaa wherein Xaa = any amino acid. 2 Pro Asn AspTrp Xaa Val Pro Lys Gly Cys Phe Met Ala 1 5 10 3 11 PRT Human PEPTIDE(2)..(10) Amino acids 2 and 10 are Xaa wherein Xaa = any amino acid. 3Pro Xaa Asp Trp Thr Val Pro Lys Gly Xaa Phe 1 5 10 4 8 PRT Human PEPTIDE(4)..(4) Amino acid 4 is Xaa wherein Xaa = any amino acid. 4 Pro Asn AspXaa Thr Val Pro Lys 1 5 5 15 PRT Human PEPTIDE (1)..(11) Amino acids 1,2 and 11 are Xaa wherein Xaa = any amino acid. 5 Xaa Xaa Ile Ala Pro GluThr Ser Glu Gly Xaa Ser Leu Gln Leu 1 5 10 15 6 10 PRT Human 6 Gly GlyTrp Pro Ile Met Val Thr Arg Lys 1 5 10 7 8 PRT Human 7 Phe Leu Ser GluGln His Gly Val 1 5 8 36 PRT Human PEPTIDE (30)..(30) Amino acid 30 isXaa wherein Xaa = any amino acid. 8 Lys Ala Met Leu Pro Leu Tyr Asp ThrGly Ser Gly Thr Ile Tyr Asp 1 5 10 15 Leu Arg His Phe Met Leu Gly IleAla Pro Asn Leu Ala Xaa Trp Asp 20 25 30 Tyr His Thr Thr 35 9 25 DNAHuman misc_feature (14)..(23) Nucleotides 14, 20 and 23 are “n” wherein“n” = any nucleotide. 9 ccgaattcaa rgcnatgytn ccnyt 25 10 26 DNA Humanmisc_feature (14)..(17) Nucleotides 14 and 17 are “n” wherein “n” = anynucleotide. 10 ccgaattcga yytnmgncay ttyatg 26 11 25 DNA Humanmisc_feature (11)..(11) Nucleotide 11 is “n” wherein “n” = anynucleotide. 11 ccggatccgt ngtrtgrtar tccca 25 12 3085 DNA Human CDS(73)..(1404) 12 tccaagctga attctcatag ctattccaaa gtctatgcac agagagccccttatcaccct 60 gatggtgtgt tt atg tcc ttt gaa ggc tac aat gtg gaa gtc cgagac aga 111 Met Ser Phe Glu Gly Tyr Asn Val Glu Val Arg Asp Arg 1 5 10gtc aag tgc ata agt ggg gtt gaa ggt gta cct tta tct aca cag tgg 159 ValLys Cys Ile Ser Gly Val Glu Gly Val Pro Leu Ser Thr Gln Trp 15 20 25 ggacct caa ggc tat ttc tac cca atc cag att gca cag tat ggg tta 207 Gly ProGln Gly Tyr Phe Tyr Pro Ile Gln Ile Ala Gln Tyr Gly Leu 30 35 40 45 agtcac tac agc aag aat cta act gaa aaa ccc cct cat ata gag gta 255 Ser HisTyr Ser Lys Asn Leu Thr Glu Lys Pro Pro His Ile Glu Val 50 55 60 tat gaaaca gca gaa gac agg gac aaa aac agc aag ccc aat gac tgg 303 Tyr Glu ThrAla Glu Asp Arg Asp Lys Asn Ser Lys Pro Asn Asp Trp 65 70 75 act gtg cccaag ggc tgc ttt atg gct agt gtg gct gat aag tca aga 351 Thr Val Pro LysGly Cys Phe Met Ala Ser Val Ala Asp Lys Ser Arg 80 85 90 ttc acc aat gttaaa cag ttc att gct cca gaa acc agt gaa ggt gta 399 Phe Thr Asn Val LysGln Phe Ile Ala Pro Glu Thr Ser Glu Gly Val 95 100 105 tcc ttg caa ctgggg aac aca aaa gat ttt att att tca ttt gac ctc 447 Ser Leu Gln Leu GlyAsn Thr Lys Asp Phe Ile Ile Ser Phe Asp Leu 110 115 120 125 aag ttc ttaaca aat gga agc gtg tct gtg gtt ctg gag acg aca gaa 495 Lys Phe Leu ThrAsn Gly Ser Val Ser Val Val Leu Glu Thr Thr Glu 130 135 140 aag aat cagctc ttc act gta cat tat gtc tca aat acc cag cta att 543 Lys Asn Gln LeuPhe Thr Val His Tyr Val Ser Asn Thr Gln Leu Ile 145 150 155 gct ttt aaagaa aga gac ata tac tat ggc atc ggg ccc aga aca tca 591 Ala Phe Lys GluArg Asp Ile Tyr Tyr Gly Ile Gly Pro Arg Thr Ser 160 165 170 tgg agc acagtt acc cgg gac ctg gtc act gac ctc agg aaa gga gtg 639 Trp Ser Thr ValThr Arg Asp Leu Val Thr Asp Leu Arg Lys Gly Val 175 180 185 ggt ctt tccaac aca aaa gct gtc aag cca aca aga ata atg ccc aag 687 Gly Leu Ser AsnThr Lys Ala Val Lys Pro Thr Arg Ile Met Pro Lys 190 195 200 205 aag gtggtt agg ttg att gcg aaa ggg aag ggc ttc ctt gac aac att 735 Lys Val ValArg Leu Ile Ala Lys Gly Lys Gly Phe Leu Asp Asn Ile 210 215 220 acc atctct acc aca gcc cac atg gct gcc ttc ttc gct gcc agt gac 783 Thr Ile SerThr Thr Ala His Met Ala Ala Phe Phe Ala Ala Ser Asp 225 230 235 tgg ctggtg agg aac cag gat gag aaa ggc ggc tgg ccg att atg gtg 831 Trp Leu ValArg Asn Gln Asp Glu Lys Gly Gly Trp Pro Ile Met Val 240 245 250 acc cgtaag tta ggg gaa ggc ttc aag tct tta gag cca ggg tgg tac 879 Thr Arg LysLeu Gly Glu Gly Phe Lys Ser Leu Glu Pro Gly Trp Tyr 255 260 265 tcc gccatg gcc caa ggg caa gcc att tct aca tta gtc agg gcc tat 927 Ser Ala MetAla Gln Gly Gln Ala Ile Ser Thr Leu Val Arg Ala Tyr 270 275 280 285 ctctta aca aaa gac cat ata ttc ctc aat tca gct tta agg gca aca 975 Leu LeuThr Lys Asp His Ile Phe Leu Asn Ser Ala Leu Arg Ala Thr 290 295 300 gcccct tac aag ttt ctg tca gag cag cat gga gtc aag gct gtg ttt 1023 Ala ProTyr Lys Phe Leu Ser Glu Gln His Gly Val Lys Ala Val Phe 305 310 315 atgaat aaa cat gac tgg tat gaa gaa tat cca act aca cct agc tct 1071 Met AsnLys His Asp Trp Tyr Glu Glu Tyr Pro Thr Thr Pro Ser Ser 320 325 330 tttgtt tta aat ggc ttt atg tat tct tta att ggg ctg tat gac tta 1119 Phe ValLeu Asn Gly Phe Met Tyr Ser Leu Ile Gly Leu Tyr Asp Leu 335 340 345 aaagaa act gca ggg gaa aaa ctc ggg aaa gaa gcg agg tcc ttg tat 1167 Lys GluThr Ala Gly Glu Lys Leu Gly Lys Glu Ala Arg Ser Leu Tyr 350 355 360 365gag cgt ggc atg gaa tcc ctt aaa gcc atg ctc ccc ttg tac gac act 1215 GluArg Gly Met Glu Ser Leu Lys Ala Met Leu Pro Leu Tyr Asp Thr 370 375 380ggc tca gga acc atc tat gac ctc cgg cac ttc atg ctt ggc att gcc 1263 GlySer Gly Thr Ile Tyr Asp Leu Arg His Phe Met Leu Gly Ile Ala 385 390 395ccc aac ctg gcc cgc tgg gac tat cac acc acc cac atc aat caa ctg 1311 ProAsn Leu Ala Arg Trp Asp Tyr His Thr Thr His Ile Asn Gln Leu 400 405 410cag ctg ctt agc acc att gat gag tcc cca atc ttc aaa gaa ttt gtc 1359 GlnLeu Leu Ser Thr Ile Asp Glu Ser Pro Ile Phe Lys Glu Phe Val 415 420 425aag agg tgg aag agc tac ctt aaa ggc agc cgg gca aag cac aac 1404 Lys ArgTrp Lys Ser Tyr Leu Lys Gly Ser Arg Ala Lys His Asn 430 435 440tagagctcag aaccaaaatc ctacgtcagc ctctgctgta cacagaaact agaggctctg 1464tgtcagcaga gcataggcac attttaaaag gctgtatact aggtttttgt ggattacatc 1524aaagtgataa atgatcctta aaaccagtct tctgagataa ttgcattcca tgggtttagt 1584gtttagaatg tcgatggcat ttatagcaga aaagtgttta gtcagtgggc tgaatgaaga 1644tgtttaactt ggcctcgctt atcaccctgt tcagttccac aggtagtcca gttctctcga 1704tttgggaaag acaatggtaa gtagctcttg atggccagct gtccagcact tgtctgaaaa 1764cttagtatgg ggctctttta aaatgtggtt atttatgttt atgttgaaag cagactttaa 1824aaaaataatg tgctaaaata cagtaaatat gtacttgtag cctgatagtg actgtgtgca 1884actttaaaaa tgatttttct tttctataaa ttaatttctt aggggtggat gagcatttgt 1944tgtgtttgtt caagttgtta tatatggaga atattttgaa tttatggttt gcttgaagtg 2004tataaattaa aaacacaacc agtgttcagg cttcacagtt atataatgta agcacaacta 2064aaatgaaact tgttgactgc acaagaaatt acaaaacaga acaaaaatgt tatctgtttt 2124atgaaactat ctacaatcag taaagatttg ataatcagta tacccctcct gtacccccat 2184tgtggtggtt tctttttgcc actatctcaa attttgtatt tcatttcaga ctacacttga 2244gagttttgtc tattttgggg ggacattttg gggacatttg ggaaatttta ctataaacct 2304agatttgatg aggaggtagt aagtttaata agcccactac cactgccttt tctagattct 2364tttccccttt aaggaaaaat attaggtcag atattataag gattgtagca gatttttttc 2424ctacttagat cattcttggt ctacagcttt ccaaactatt gatgtacaca aaatacatag 2484tttttgtgta agctttcaaa cttttctggt gttttttctt tgcagttttt aattttaaat 2544tatttcagct cttggataaa agtgatgcta ctatattagc tgtacatgtg taatcagacc 2604tttattttgg ttttatatcc cacatacctc acataaatag gcatcatagc cctcacaccc 2664tgggcagtgt ctgctctagg acttaggcag taggtcagaa ctgagggagg ttgattttgc 2724tgtctctgtt ttagtgtatg acaatacagt aaatcaatac aataacttat acagattgga 2784aatacgagat ccggtacttt cagaggactg agtctgacac acgcagtgca gtgtgtgtgt 2844gacctgtatg aaatgcacat caagagcgag gtggcacctg cctgccactg catcttgcct 2904ggacttagtc taccaacacc actcagaaat ggcaaaatgc atacatgcct ttgagcaaca 2964tatatgttgt atcagcagcc ggaacgaaga cctacaactg acatgaaact gttagtcact 3024aagtcgtgtc caactctttg tgacctcata gactgtagcc cgccaggctt ctttgtccat 3084 g3085 13 444 PRT Human 13 Met Ser Phe Glu Gly Tyr Asn Val Glu Val Arg AspArg Val Lys Cys 1 5 10 15 Ile Ser Gly Val Glu Gly Val Pro Leu Ser ThrGln Trp Gly Pro Gln 20 25 30 Gly Tyr Phe Tyr Pro Ile Gln Ile Ala Gln TyrGly Leu Ser His Tyr 35 40 45 Ser Lys Asn Leu Thr Glu Lys Pro Pro His IleGlu Val Tyr Glu Thr 50 55 60 Ala Glu Asp Arg Asp Lys Asn Ser Lys Pro AsnAsp Trp Thr Val Pro 65 70 75 80 Lys Gly Cys Phe Met Ala Ser Val Ala AspLys Ser Arg Phe Thr Asn 85 90 95 Val Lys Gln Phe Ile Ala Pro Glu Thr SerGlu Gly Val Ser Leu Gln 100 105 110 Leu Gly Asn Thr Lys Asp Phe Ile IleSer Phe Asp Leu Lys Phe Leu 115 120 125 Thr Asn Gly Ser Val Ser Val ValLeu Glu Thr Thr Glu Lys Asn Gln 130 135 140 Leu Phe Thr Val His Tyr ValSer Asn Thr Gln Leu Ile Ala Phe Lys 145 150 155 160 Glu Arg Asp Ile TyrTyr Gly Ile Gly Pro Arg Thr Ser Trp Ser Thr 165 170 175 Val Thr Arg AspLeu Val Thr Asp Leu Arg Lys Gly Val Gly Leu Ser 180 185 190 Asn Thr LysAla Val Lys Pro Thr Arg Ile Met Pro Lys Lys Val Val 195 200 205 Arg LeuIle Ala Lys Gly Lys Gly Phe Leu Asp Asn Ile Thr Ile Ser 210 215 220 ThrThr Ala His Met Ala Ala Phe Phe Ala Ala Ser Asp Trp Leu Val 225 230 235240 Arg Asn Gln Asp Glu Lys Gly Gly Trp Pro Ile Met Val Thr Arg Lys 245250 255 Leu Gly Glu Gly Phe Lys Ser Leu Glu Pro Gly Trp Tyr Ser Ala Met260 265 270 Ala Gln Gly Gln Ala Ile Ser Thr Leu Val Arg Ala Tyr Leu LeuThr 275 280 285 Lys Asp His Ile Phe Leu Asn Ser Ala Leu Arg Ala Thr AlaPro Tyr 290 295 300 Lys Phe Leu Ser Glu Gln His Gly Val Lys Ala Val PheMet Asn Lys 305 310 315 320 His Asp Trp Tyr Glu Glu Tyr Pro Thr Thr ProSer Ser Phe Val Leu 325 330 335 Asn Gly Phe Met Tyr Ser Leu Ile Gly LeuTyr Asp Leu Lys Glu Thr 340 345 350 Ala Gly Glu Lys Leu Gly Lys Glu AlaArg Ser Leu Tyr Glu Arg Gly 355 360 365 Met Glu Ser Leu Lys Ala Met LeuPro Leu Tyr Asp Thr Gly Ser Gly 370 375 380 Thr Ile Tyr Asp Leu Arg HisPhe Met Leu Gly Ile Ala Pro Asn Leu 385 390 395 400 Ala Arg Trp Asp TyrHis Thr Thr His Ile Asn Gln Leu Gln Leu Leu 405 410 415 Ser Thr Ile AspGlu Ser Pro Ile Phe Lys Glu Phe Val Lys Arg Trp 420 425 430 Lys Ser TyrLeu Lys Gly Ser Arg Ala Lys His Asn 435 440

1. An isolated or recombinant DNA sequence coding for a mammalian,including human, glucuronyl C5-epimerase or a functional derivativethereof capable of converting D-glucuronic acid (GlcA) to L-iduronicacid (IdoA).
 2. A DNA sequence according to claim 1 constituted by anucleotide sequence comprising nucleotide residues 1 to 1404, inclusive,as depicted in the sequence listing.
 3. A DNA sequence according toclaim 2 constituted by a nucleotide residue comprising nucleotideresidues 73 to 1404, inclusive, as depicted in the sequence listing. 4.A DNA sequence according to claim 2 constituted by a nucleotide residuecomprising nucleotide residues 1 to 1404, inclusive, as depicted in thesequence listing.
 5. A recombinant expression vector containing atranscription unit comprising a DNA sequence according to any one of thepreceding claims, a transcriptional promoter, and a polyadenylationsequence.
 6. A host cell transformed with the recombinant expressionvector of claim
 5. 7. A process for the manufacture of a glucuronylC5-epimerase or a functional derivative thereof capable of convertingD-glucuronic acid (GlcA) to L-iduronic acid (IdoA), comprisingcultivation of a cell line transformed with a recombinant expressionvector according to claim 5 in a nutrient medium allowing expression andsecretion of said epimerase or functional derivative thereof.
 8. Aglucuronyl C5-epimerase or a functional derivative thereof wheneverprepared by the process of claim 7.